Various processes for producing nucleotides modified on the phosphate residue have already been described in the past. A review of currently used techniques to synthesize nucleoside triphosphates (NTP) may be found in Burgess, K., and Cook, D., Chem. Rev. 100 (2000) 2047-2059 and a review especially of NTPs with a modified triphosphate side chain may be found in Koukhareva, Vaghefi and Lebedev, Nucleoside Triphosphates and their Analogs (2005) Chapter 2, “Synthesis and properties of NTP analogs with modified Triphosphate side chains”, Ed. M. Vaghefi, CRC Press, Taylor & Francis, Boca Raton. Triphosphates are of outstanding importance especially because, after cleavage of the pyrophosphate, they are incorporated as monophosphate substrates into long chain nucleic acids by DNA or RNA polymerases. Usually nowadays nucleoside monophosphates are firstly prepared which are subsequently enzymatically converted into triphosphates for example by kinases.
Modified nucleoside triphosphates are advantageous for various applications such as for example the preparation of aptamers (WO 03/50290) or antisense molecules (U.S. Pat. No. 5,587,361) and they are also used in sequencing (WO 02/46468) and PCR (WO 03/27258). Use of alpha-thio-dATP as a substrate for pyrosequencing reactions has become particularly important (WO 05/54431).
Alpha-phosphate-modified nucleoside triphosphates such as for example alpha thio-triphosphates or alpha-amino NTPs (Simoncsits, A., Tomasz, J., A new type of nucleoside 5′-triphosphate analog: P1-(nucleoside 5′-) P1-aminotriphosphates. Tetrahedron Letters 17(44) (1976) 3995-8) have been known for a long time. The preparation of alpha-methyl phosphonates (JP 01224392) and alpha-borano phosphates (Tomasz, J., et al., 5′-P-borane-substituted thymidine phosphate and triphosphate, Angewandte Chemie 104(10) (1992) 1404-6; Tomasz, J., et al., Angew. Chemie 31(10) (1992) 1373-5) has also already been described. WO 03/008432 describes the separation of N-alkyl-substituted derivatives by treating nucleotidyl-cyclo-triphosphite with an oxidizing agent in the presence of amines.
The preparation of modified nucleotide esters with the aid of azides is also known from the prior art. Baschang and Kvita, Angewandte Chemie 85(1) (1973) 43-44 describe the reaction of a nucleotide phosphoric acid triester with azides such as methyl-sulfonyl azide to prepare trialkyl(aryl)-imidophosphates. However, they are unstable and decay.
Nielsen, J., and Caruthers, M. H., J. Am. Chem. Soc. 110 (1988) 6275-6276 describe the reaction of deoxynucleoside phosphites provided with a 2-cyano-1,1-dimethylethyl protecting group in the presence of alkyl azide. In addition the authors suggest that this principle is suitable for preparing oligonucleotides with a modified inter-nucleoside phosphate without elucidating which types of modifications prepared with the aid of the disclosed process could have special advantages. In particular the authors propose introducing alkyl residues into oligonucleotides during oligo-nucleotide synthesis. Nucleoside triphosphates are not a subject matter of this publication.
Thus, all these publications describe the preparation of molecules which contain a phosphoramidite instead of a phosphate residue. However, molecules containing phosphoramidites are subject to hydrolysis because the amine group is protonated in an acidic medium and is then substituted by water. Consequently the previously described methods are of only very limited suitability for producing stable alpha-phosphate-modified nucleotides.
The technical object forming the basis of the present invention was thus to provide improved nucleotides modified on the alpha-phosphate, so-called alpha-phosphate mimetics, and to provide simple processes for their production.